Analysis of critical protein–protein interactions of SARS-CoV-2 capping and proofreading molecular machineries towards designing dual target inhibitory peptides

In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein–protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues’ interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14’s exoribonuclease (ExoN) and nsp16’s 2′O-methyltransferase (2′O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2′O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell’s innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein–protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide–protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors.


Figure S5
: Secondary structure plot of the SARS-CoV-2 nsp14-nsp10 complex. The red boxes denote the predicted interfacial residues. The predicted hotspot residues are indicated with red asterisks. Figure S6: Analysis of the SARS-CoV nsp16-nsp10 protein-protein interface and hotspot residues. The interfaces and hotspots were mapped to the structures at the protein-protein interface of nsp16 (gray) and nsp10 (cyan), which are colored orange and red, respectively. The hotspot residues are shown in red labels. Figure S7: Analysis of the MERS-CoV nsp16-nsp10 protein-protein interface and hotspot residues. The interfaces and hotspots were mapped to the structures at the protein-protein interface of nsp16 (gray) and nsp10 (cyan), which are colored orange and red, respectively. The hotspot residues are shown in red labels. Figure S8: Analysis of the HCoV-OC43 nsp16-nsp10 protein-protein interface and hotspot residues. The interfaces and hotspots were mapped to the structures at the protein-protein interface of nsp16 (gray) and S10 nsp10 (cyan), which are colored orange and red, respectively. The hotspot residues are shown in red labels.

Figure S9:
The results of computational alanine scanning (CAS) calculations for the predicted key interacting residues of the SARS-CoV-2 nsp16-nsp10 and nsp14-nsp10 complexes. (a) the bar chart summarized the changes in binding affinity upon mutation of nsp10 key residues to alanine in the nsp14-nsp10 complex, (b) the bar chart summarized the changes in binding affinity upon mutation of nsp14 key residues to alanine in the nsp14-nsp10 complex, (c) the bar chart summarized the changes in binding affinity upon mutation of nsp10 key residues to alanine in the nsp16-nsp10 complex, and (d) the bar chart S11 summarized the changes in binding affinity upon mutation of nsp16 key residues to alanine in the nsp16-nsp10 complex. The red and blue bars show the decreasing (negative ΔΔG Affinity ) and increasing (positive ΔΔG Affinity ) impacts on PPIs, respectively.

Figure S10
: Interactions between wild-type (V42, L45, and Y96 of nsp10) and mutant (alanine) residues and their nearby residues in the nsp16-nsp10 complex. The interactions between the wild-type and mutant residues and their nearby residues on the left, and the inter-residue interactions of the wild-type and mutant on the right for: (a) V42 of nsp10 mutated to alanine in the nsp16-nsp10 complex, (b) L45 of nsp10 mutated to alanine in the nsp16-nsp10 complex, and (c) Y96 of nsp10 mutated to alanine in the nsp16-nsp10 complex. The interactions of wild type and mutant residues are represented by blue and S12 purple boxes, respectively. The nsp16 protein is chain A and is represented in cartoon and in blue. The nsp10 protein is chain B depicted in cartoon and red. The dashed lines and straight lines represent the wild type and mutant inter-residues interactions, respectively. The aromatic, carbonyl, hydrophobic, hydrogen bond, ionic, VDW, ring-ring, and polar-Hbond interactions are colored in light green, dark blue, dark green, red, yellow, light blue, black, and orange, respectively.

Figure S11
: Interactions between wild-type (V84, D106, and V44 of nsp16) and mutant (alanine) residues and their nearby residues in the nsp16-nsp10 complex. The interactions between the wild-type and mutant residues and their nearby residues on the left, and the inter-residue interactions of the wildtype and mutant on the right for: (a) V84 of nsp16 mutated to alanine in the nsp16-nsp10 complex, (b) D106 of nsp16 mutated to alanine in the nsp16-nsp10 complex, (c) V44 of nsp16 mutated to alanine in the nsp16-nsp10 complex. The interactions of wild type and mutant residues are represented by blue and purple boxes, respectively. The nsp16 protein is chain A and is represented in cartoon and in blue. The nsp10 protein is chain B depicted in cartoon and red. The dashed lines and straight lines represent the wild type and mutant inter-residues interactions, respectively. The aromatic, carbonyl, hydrophobic, hydrogen bond, ionic, VDW, ring-ring, and polar-Hbond interactions are colored in light green, dark blue, dark green, red, yellow, light blue, black, and orange, respectively.

Figure S12:
Interactions between wild-type (F16, H80, and Y96 of nsp10) and mutant (alanine) residues and their nearby residues in the nsp14-nsp10 complex. The interactions between the wild-type and mutant residues and their nearby residues on the left, and the inter-residue interactions of the wild-type and mutant on the right for: (a) F16 of nsp10 mutated to alanine in the nsp14-nsp10 complex, (b) H80 of nsp10 mutated to alanine in the nsp14-nsp10 complex, and (c) Y96 of nsp10 mutated to alanine in the nsp14-nsp10 complex. The interactions of wild type and mutant residues are represented by blue and purple boxes, respectively. The nsp14 protein is chain B and is represented in cartoon and blue. The nsp10 protein is chain A depicted in cartoon and red. The dashed lines and straight lines represent the wild type and mutant inter-residues interactions, respectively. The aromatic, carbonyl, hydrophobic, hydrogen bond, ionic, VDW, ring-ring, and polar-Hbond interactions are colored in light green, dark blue, dark green, red, yellow, light blue, black, and orange, respectively.

Figure S13:
Interactions between wild-type (F8, P24, and F60 of nsp14) and mutant (alanine) residues and their nearby residues in the nsp14-nsp10 complex. The interactions between the wild-type and mutant residues and their nearby residues on the left, and the inter-residue interactions of the wild-type and mutant on the right for: (a) F8 of nsp14 mutated to alanine in the nsp14-nsp10 complex, (b) P24 of nsp14 mutated to alanine in the nsp14-nsp10 complex, and (c) F60 of nsp14 mutated to alanine in the nsp14-nsp10 complex. The interactions of wild type and mutant residues are represented by blue and purple boxes, respectively. The nsp14 protein is chain B and is represented in cartoon and blue. The nsp10 protein is chain A depicted in cartoon and red. The dashed lines and straight lines represent the wild type and mutant inter-residues interactions, respectively. The aromatic, carbonyl, hydrophobic, hydrogen bond, ionic, VDW, ring-ring, and polar-Hbond interactions are colored in light green, dark blue, dark green, red, yellow, light blue, black, and orange, respectively.

Figure S19:
The heat map for residue-residue pairwise interaction energies (IEs) between the predicted key interacting residues of nsp10 and nsp16 in the SAR-CoV-2 nsp16-nsp10 complex. The negative (with the stabilizing roles in PPIs) and positive (with the destabilizing roles in PPIs) pairwise IEs are colored in red and blue, respectively.

Figure S20:
The heat map for residue-residue pairwise interaction energies (IEs) between the predicted key interacting residues of nsp10 and nsp14 in the SARS-CoV-2 nsp14-nsp10 complex. The negative (with the stabilizing roles in PPIs) and positive (with the destabilizing roles in PPIs) pairwise IEs are colored in red and blue, respectively.

Figure S21
: Node centrality parameters in the network, including degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) of the predicted key interacting residues of the SARS-CoV-2 nsp16-nsp10 complex. (a) Node centrality parameters for the predicted key interacting residues of nsp10, and (b) the node centrality parameters for the predicted key interacting residues of nsp16. Figure S22: Node centrality parameters in the network, including degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) of the predicted key interacting residues of the SARS-CoV-2 nsp14-nsp10 complex. (a) Node centrality parameters for the predicted key interacting residues of nsp10, and (b) the node centrality parameters for the predicted key interacting residues of nsp14.

Figure S28
: Two-dimensional residue interaction maps of OLP-16 and OLP-17 peptides and the targets. (a) OLP-16 and OLP-17 peptide-protein interactions when target was nsp16, and (b) OLP-16 and OLP-17 peptide-protein interactions when target was nsp14. The black dashed lines and brown arcs with spokes represent the hydrogen bonds and the hydrophobic contacts, respectively.